Lipid-based nanoformulations for peptide delivery

Nanoformulations have attracted a lot of attention because of their size-dependent properties. Among the array of nanoformulations, lipid nanoformulations (LNFs) have evoked increasing interest because of the advantages of their high degree of biocompatibility and versatility. The performance of lipid nanoformulations is greatly influenced by their composition and structure. Therapeutic peptides represent a growing share of the pharmaceutical market. However, the main challenge for their development into commercial products is their inherent physicochemical and biological instability. Important peptides such as insulin, calcitonin and cyclosporin A have been incorporated into LNFs. The association or encapsulation of peptides within lipid-based carriers has shown to protect the labile molecules against enzymatic degradation. This review describes strategies used for the formulation of peptides and some methods used for the assessment of association efficiency. The advantages and drawbacks of such carriers are also described

Slow-Release Formulation of Cowpea Mosaic Virus for In Situ Vaccine Delivery to Treat Ovarian Cancer.

The plant viral nanoparticle cowpea mosaic virus (CPMV) is shown to be an effective immunotherapy for ovarian cancer when administered as in situ vaccine weekly, directly into the intraperitoneal (IP) space in mice with disseminated tumors. While the antitumor efficacy is promising, the required frequency of administration may pose challenges for clinical implementation. To overcome this, a slow release formulation is developed. CPMV and polyamidoamine generation 4 dendrimer form aggregates (CPMV-G4) based on electrostatic interactions and as a function of salt concentration, allowing for tailoring of aggregate size and release of CPMV. The antitumor efficacy of a single administration of CPMV-G4 is compared to weekly administration of soluble CPMV in a mouse model of peritoneal ovarian cancer and found to be as effective at reducing disease burden as more frequent administrations of soluble CPMV; a single injection of soluble CPMV, does not significantly slow cancer development. The ability of CPMV-G4 to control tumor growth following a single injection is likely due to the continued presence of CPMV in the IP space leading to prolonged immune stimulation. This enhanced retention of CPMV and its antitumor efficacy demonstrates the potential for viral-dendrimer hybrids to be used for delayed release applications

Extracellular membrane vesicles from Limosilactobacillus reuteri strengthen the intestinal epithelial integrity, modulate cytokine responses and antagonize activation of TRPV1

Bacterial extracellular membrane vesicles (MV) are potent mediators of microbe-host signals, and they are not only important in host-pathogen interactions but also for the interactions between mutualistic bacteria and their hosts. Studies of MV derived from probiotics could enhance the understanding of these universal signal entities, and here we have studied MV derived from Limosilactobacillus reuteri DSM 17938 and BG-R46. The production of MV increased with cultivation time and after oxygen stress. Mass spectrometry-based proteomics analyses revealed that the MV carried a large number of bacterial cell surface proteins, several predicted to be involved in host-bacteria interactions. A 5 '-nucleotidase, which catalyze the conversion of AMP into the signal molecule adenosine, was one of these and analysis of enzymatic activity showed that L. reuteri BG-R46 derived MV exhibited the highest activity. We also detected the TLR2 activator lipoteichoic acid on the MV. In models for host interactions, we first observed that L. reuteri MV were internalized by Caco-2/HT29-MTX epithelial cells, and in a dose-dependent manner decreased the leakage caused by enterotoxigenic Escherichia coli by up to 65%. Furthermore, the MV upregulated IL-1 beta and IL-6 from peripheral blood mononuclear cells (PBMC), but also dampened IFN-gamma and TNF-alpha responses in PBMC challenged with Staphylococcus aureus. Finally, we showed that MV from the L. reuteri strains have an antagonistic effect on the pain receptor transient receptor potential vanilloid 1 in a model with primary dorsal root ganglion cells from rats. In summary, we have shown that these mobile nanometer scale MV reproduce several biological effects of L. reuteri cells and that the production parameters and selection of strain have an impact on the activity of the MV. This could potentially provide key information for development of innovative and more efficient probiotic products

Correlation between the antimicrobial activity and metabolic profiles of cell free supernatants and membrane vesicles produced by lactobacillus reuteri DSM 17938

The aim of the work is to assess the antimicrobial activities of Cell Free Supernatants (CFS) and Membrane Vesicles (MVs), produced by Lactobacillus reuteri DSM 17938, versus Gram-positive and Gram-negative bacteria and investigate their metabolic profiles. The Minimum Inhibitory Concentration was determined through the broth microdilution method and cell proliferation assay and the Minimum Bactericidal Concentration was determined by Colony Forming Units counts. The characteristics of the antimicrobial compounds were evaluated by pH adjustments, proteinase treatment, and size fractionation of the CFS. The cytotoxicity of CFS was tested on two human cell lines. A detailed snapshot of the L. reuteri metabolism was attained through an untargeted metabolic profiling by means of high resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) coupled with Electrospray Ionization Source (ESI). The results showed (i) a greater efficacy of CFS and its fractions towards Gram-negative compared to Gram-positive bacteria; (ii) an antimicrobial effect related to pH-dependent compounds but not to MVs; (iii) a molecular weight < 3 KDa as well as an a non-proteinaceous nature of the antimicrobial compounds; and (iv) more than 200 and 500 putative metabolites annotated in MVs and supernatants, covering several classes of metabolites, including amino acids, lipids, fatty and organic acids, polyalcohols, nucleotides, and vitamins. Some putative compounds were proposed not only as characteristic of specific fractions, but also possibly involved in antimicrobial activity

Correlation between the Antimicrobial Activity and Metabolic Profiles of Cell Free Supernatants and Membrane Vesicles Produced by Lactobacillus reuteri DSM 17938

The aim of the work is to assess the antimicrobial activities of Cell Free Supernatants (CFS) and Membrane Vesicles (MVs), produced by Lactobacillus reuteri DSM 17938, versus Gram-positive and Gram-negative bacteria and investigate their metabolic profiles. The Minimum Inhibitory Concentration was determined through the broth microdilution method and cell proliferation assay while the Minimum Bactericidal Concentration was determined by Colony Forming Units counts. The characteristics of the antimicrobial compounds were evaluated by pH adjustments, proteinase treatment, and size fractionation of the CFS. The cytotoxicity of CFS was tested on two human cell lines. A detailed snapshot of the L. reuteri metabolism was attained through an untargeted metabolic profiling by means of high resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) coupled with Electrospray Ionization Source (ESI). The results showed (i) a greater efficacy of CFS and its fractions towards Gram-negative compared to Gram-positive bacteria; (ii) an antimicrobial effect related to pH-dependent compounds but not to MVs; (iii) a molecular weight < 3 KDa as well as an a non-proteinaceous nature of the antimicrobial compounds; and (iv) more than 200 and 500 putative metabolites annotated in MVs and supernatants, covering several classes of metabolites, including amino acids, lipids, fatty and organic acids, polyalcohols, nucleotides, and vitamins. Some putative compounds were proposed not only as characteristic of specific fractions, but also possibly involved in antimicrobial activity

Peptide interactions with bacterial lipopolysaccharides

Peptide and protein interactions with (lipo)polysaccharides are important in various biological contexts, including lipoprotein deposition at proteoglycan-covered endothelial surfaces in atherosclerosis, lectin functionality, and the interaction of antimicrobial and anti-inflammatory peptides and proteins with (lipo)polysaccharides. The latter of these areas, which is the topic of this review, has attracted considerable interest during the last few years, since antimicrobial peptides may offer novel therapeutic opportunities in an era of growing problems with antibiotic resistance, and persisting problems with both acute and chronic inflammation. In the present overview, physicochemical factors affecting peptide interactions with bacterial (lipo)polysaccharides are discussed, both in solution and at membrane interfaces. In doing so, an attempt is made to illustrate how physicochemical factors affect the antimicrobial and anti-endotoxic functionality of such peptides, and how knowledge on this can be translated into therapeutic opportunities, e.g., in sepsis. (C) 2013 Elsevier Ltd. All rights reserved

Fabrication of tailorable pH responsive cationic amphiphilic microgels on a microfluidic device for drug release

Cationic, amphiphilic microgels of differing compositions based on hydrophilic, pH, and thermoresponsive 2-(dimethylamino)ethyl methacrylate (DMAEMA) and hydrophobic, nonionic n-butyl acrylate (BuA) are synthesized using a lab-on-a-chip device. Hydrophobic oil-in-water (o/w) droplets are generated via a microfluidic platform, with the dispersed (droplet) phase containing the DMAEMA and BuA, alongside the hydrophobic cross-linker, ethylene glycol dimethacrylate, and a free radical initiator in an organic solvent. Finally, the hydrophobic droplets are photopolymerized via a UV light source as they traverse the microfluidic channel to produce the cationic amphiphilic microgels. This platform enables the rapid, automated, and in situ production of amphiphilic microgels, which do not match the core-shell structure of conventionally prepared microgels but are instead based on random amphiphilic copolymers of DMAEMA and BuA between the hydrophobic cross-links. The microgels are characterized in terms of their swelling and encapsulation abilities, which are found to be influenced by both the pH response and the hydrophobic content of the microgels. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018, 56, 59–66

Interactions of NIPAM nanogels with model lipid multi-bilayers: A neutron reflectivity study

The authors wish to thank the ISIS, STFC, UK, for beam time on SURF and INTER (RB 1410123). The European Commission (FP7 Marie Curie Actions, projects NANODRUG (MCITN-2011-289554) and NANOLEM (PIEF-GA-2013-627146)), the Chinese Scholarship Council and Queen Mary, University of London are gratefully acknowledged for financial support

Sälja fastigheter eller aktier? - När verksamhetens inriktning hindrar att andelar blir näringsbetingade

Under 2003 infördes nya regler som gjorde utdelning och försäljning av näringsbetingade andelar skattebefriade. Genom att bolagsförpacka en fastighet innan försäljning kunde bolagen undkomma kapitalvinstbeskattning om andelarna i dotterbolaget var näringsbetingade. Om andelarna i dotterbolaget däremot är att klassificera som lagertillgångar kommer det säljande bolaget att beskattas för kapitalvinsten. Det är därför av stor vikt att det säljande utreder vilken karaktär andelarna i dotterbolaget kommer att få vid försäljningstidpunkten, då syftet med paketering är att försäljningen skall bli skattefri.Paketeringsförandet i sin enklaste form fungerar så att moderbolaget startar ett helägt dotterbolag. Moderbolaget genomför sedan en underprisöverlåtelse av fastigheten till dotterbolaget. Aktierna i dotterbolaget avyttras till en extern köpare för marknadspris.Syftet med uppsatsen är att undersöka vilken typ av bolagsverksamhet som styr hur andelarna i ett dotterbolag klassificeras. Grundregeln säger att om ett moderbolag är verksamma inom handel med fastigheter, byggnads- eller tomtrörelse kommer andelarna direkt att klassificeras som lagertillgångar.Jag har i uppsatsen kommit fram till att det i förarbetena inte finns någon tydlig definition om vad som är handel med fastigheter, utan vägledning i detta har fått göras utifrån praxis. Enligt två nya avgöranden tyder det på att upp till sexton fastigheter kan säljas utan att bolaget skall anses bedriva handel med fastigheter. Lagen är tydlig i att andelar som är förknippade med byggnadsrörelsen omöjliggör att andelar kan vara näringsbetingade. Jag har studerat hur stor anknytningen till byggnadsrörelse fysisk person eller fastighet kan ha utan att bli byggmästarsmittade. I praxis har jag funnit att den fysiska personens bestämmande över byggnadsrörelse är något som gör reglerna i 27. kap. IL. gällande. För ett fastighet är det anknytning till byggnadsrörelsen som gör dito. Dock finns möjligheten för ett moderbolag som driver byggnadsrörelse att skapa ett holdingbolag som i sin tur äger det dotterbolag som skall säljas. Genom detta skapas en distans till byggnadsrörelsen som gör att andelarna i dotterbolaget kan vara näringsbetingade.In 2003, new rules were introduced for taxable dividends and sales of business-related shares. It made it possible to sell properties without any taxation, by "packing" the property in an affiliate company before its introduced to the market. If the affiliate company is to classify as business-related shares, the disposal will be without capital gain tax, if the shares in the affiliate company is storage assets the company will be taxed for its earnings. It is therefore important that the selling company will investigate the nature of the shares that the affiliate will have, since the purpose of packaging is that the sale will be out taxation.The process of "packing", in its simplest form is when that a parent company creates a wholly owned subsidiary and later carries out an undervalued transfer of the property to the subsidiary. The shares of the subsidiary are then sold to an outside buyer to the market price.The purpose of this essay is to investigate which activities of a parent company that controls the classification of the shares in the subsidiary company. The general rule stipulates that if parent company is affiliated with business regarding properties, lots or construction the shares will be directly classified as "inventory assets".This thesis concludes that the preparatory legislative documents does not give a clear definition of what is considered trade in real estate or construction business. Guidance in this matter has thus been completed trough court practice. According to two new rulings the sale of up to sixteen properties can be carried out without the company necessary being classified as executing business with properties. What controls, if a company's operation as a "construction business" shall make it impossible that the shares in a subsidiary may be business-related, is according to practice , control of the company for a physical person and the connection between a "construction business" and the real estate keeping company. However, there is a possibility of distancing construction operations from a subsidiary, that is to be sold, by having the subsidiary company being owned by a holding company. Thus circumventing the relation to the "construction business" that makes the shares "inventory assets"